Journal: The EMBO Journal

Article Title: Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells

doi: 10.15252/embj.2019101828

Figure Lengend Snippet: mTEC promiscuously expressing TSPAN8 and/or GP2 (upper right panel/red) on their cell surface can be identified by flow cytometry (only final gates are shown: see Appendix Fig S1 for full gating strategy). mTEC were identified as CD45 − EpCAM + Ly51 − ( Appendix Fig S1 ) and the gates for TSPAN8/GP2 were set against isotype control antibodies (left panels/grey). Lower right panel: bar graph showing mean frequency (±SD) of TSPAN8 + , GP2 + , and TSPAN8 + GP2 + cells within total mTEC; results represent pooled data from 3 (TSPAN8 + ), 4 (GP2 + ) and 2 (TSPAN8 + GP2 + ) independent experiments each containing three individual mice. Identification of TSPAN8 or GP2 protein expression via FACS reflects mRNA expression. Bar graph showing mean expression (±SD) of Tspan8 and Gp2 mRNA relative to β‐actin by RT–qPCR on FACS sorted mTEC negative or positive for TSPAN8 or GP2 protein, respectively; n = 3, representative of two independent experiments. Significance by Students t ‐test; *** P < 0.001. Schematic representation of cell populations sorted by flow cytometry for single‐cell RNA‐sequencing. Numbers of recovered cells are indicated below, coloured by category. t‐SNE visualisation of mTEC subpopulations from all experiments coloured by surface phenotype established via flow cytometry; see Fig B and C for the distributions of unselected mTEC.

Article Snippet: After staining, cells were acquired and sorted using a FACS Aria III (BD Biosciences) and analysed using FlowJo v10 and GraphPad Prism 7; statistical analyses were performed using a t ‐test with Bonferroni correction for multiple comparisons where appropriate; differences were considered significant if the adjusted P ‐value was ≤ 0.05.

Techniques: Expressing, Flow Cytometry, Quantitative RT-PCR, RNA Sequencing Assay